Tissue-specific in vivo transformation of plasmid DNA in Neotropical tadpoles using electroporation

Electroporation is an increasingly common technique used for exogenous gene expression in live animals, but protocols are largely limited to traditional laboratory organisms. The goal of this protocol is to test in vivo electroporation techniques in a diverse array of tadpole species. We explore electroporation efficiency in tissue-specific cells of five species from across three families of tropical frogs: poison frogs (Dendrobatidae), cryptic forest/poison frogs (Aromobatidae), and glassfrogs (Centrolenidae). These species are well known for their diverse social behaviors and intriguing physiologies that coordinate chemical defenses, aposematism, and/or tissue transparency. Specifically, we examine the effects of electrical pulse and injection parameters on species- and tissue-specific transfection of plasmid DNA in tadpoles. After electroporation of a plasmid encoding green fluorescent protein (GFP), we found strong GFP fluorescence within brain and muscle cells that increased with the amount of DNA injected and electrical pulse number. We discuss species-related challenges, troubleshooting, and outline ideas for improvement. Extending in vivo electroporation to non-model amphibian species could provide new opportunities for exploring topics in genetics, behavior, and organismal biology.

ABSTRACT Electroporation is an increasingly common technique used for exogenous gene expression in live animals, but protocols are largely limited to traditional laboratory organisms. The goal of this protocol is to enable in vivo electroporation techniques in a diverse array of tadpole species. We explore electroporation efficiency in tissuespecific cells of five species from across three families of tropical frogs-poison frogs (Dendrobatidae), forest frogs (Aromobatidae), and glassfrogs (Centrolenidae). These species are well-known for their diverse social behaviors and intriguing physiologies that coordinate chemical defenses, aposematism, and/or transparency.
Specifically, we examine the effects of electrical pulse and injection parameters on species-and tissue-specific transfection of plasmid DNA in tadpoles. After electroporation of a plasmid encoding green fluorescent protein (GFP), we found strong GFP fluorescence within brain and muscle cells that increases with the amount of DNA injected and electrical pulse number. We discuss species-related challenges, troubleshooting, and outline ideas for improvement. Extending in vivo electroporation to diverse amphibian species will offer a powerful approach to explore topics in genetics, behavior, and organismal biology. Remove the tips from two 5 mL serological pipettes using scissors 4 Solder two ~ 5 mm X 8 mm pieces of platinum foil to separate electrical lead wires to make an electrode 5 Run one electrode wire through each cut serological pipette and secure it with electrical tape 6 Construct a platform out of clay evenly spread over the top of a Petri dish 7

ATTACHMENTS
Embed the anode into the clay with the foil exposed near the center of the Petri dish 8 Create a tadpole-sized (~ 1 cm) impression adjacent the anode foil   Set the stimulator parameters to 10 pps, 0.1 ms delay, 15 ms duration, and 30 V

Note
These settings are a suggested starting point. Optimizing these parameters for each species, as the best pulse shape and settings may vary, is recommended.
Pull glass capillaries using a pipette puller

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Break the pipette tip at an angle using forceps to create a beveled tip

31.2
For experiments targeting brain cells, place the tadpole dorsal side up with its head in the depression

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Orient the platform such that the head of the tadpole is facing toward the injector

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Lower the injector and insert the pipette into the target tissue

33.1
For experiments targeting muscle fibers, insert the pipette into a tail myomere

33.2
For experiments targeting brain cells, insert the pipette into the brain ventricle

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Inject the plasmid DNA with a 5-10 s interval between each injection

34.1
For experiments targeting muscle fibers, deliver two injections

34.2
For experiments targeting brain cells, deliver three injections

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Remove the pipette from the tadpole

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Orient the platform such that the head of the tadpole is facing toward the electrode

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Lower the electrode until it is in full contact with the target tissue

37.1
For experiments targeting muscle fibers, the tail should lay on top of the anode and the cathode should press on the tail directly above the anode

37.2
For experiments targeting brain cells, the electrode should be touching the head on either side of the brain 1m 2m 2m 1m 1m 5m Figure 7. Brain electroporation in a Ranitomeya imitator tadpole with the electrode making contact with either side of the brain.

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Deliver the electrical pulses

Note
The pulse range for targeting brain cells is a suggested starting point. Optimizing the protocol for species-specific applications to maximize transfection efficiency is recommended.

38.1
For experiments targeting muscle fibers, deliver 4-8 double pulses with a 1 s interval between each set of pulses

38.2
For experiments targeting brain cells, deliver 4-10 pulses with a 1 s interval between each pulse

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Transfer the tadpole to fresh tadpole water for several hours to recover 2m 4h Roughly 24 to 48 hours after electroporation, screen tadpoles for plasmid uptake by imaging GFP-positive cells

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Anesthetize the tadpole by placing it in a Petri dish of room temperature 0.03% MS-222 for 5 minutes

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Move the tadpole to a new Petri dish with tadpole water and place under a stereomicroscope with a GFP filter

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Center the imaging field on the target tissue and capture the fluorescent image

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Transfer the tadpole to fresh tadpole water for several hours to recover 5m 5m 10m 4h In Vivo Screening 20m